Cancer is a major public health problem worldwide. In the United States alone, approximately 560,000 people died of cancer in 2006. See, e.g., U.S. Mortality Data 2006, National Center for Health Statistics, Centers for Disease Control and Prevention (2009). Many types of cancer have been described in the medical literature. Examples include cancer of the blood, bone, skin, lung, colon, breast, prostate, ovary, brain, kidney, bladder, pancreas, and liver. The incidence of cancer continues to climb as the general population ages and as new forms of cancer develop. A continuing need exists for effective therapies to treat subjects with cancer.
Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic cell disorders. MDS affect approximately 40,000-50,000 people in the U.S. and 75,000-85,000 people in Europe. MDS may be characterized by a cellular marrow with impaired morphology and maturation (dysmyelopoiesis), peripheral blood cytopenias, and a variable risk of progression to acute leukemia, resulting from ineffective blood cell production. See, e.g., The Merck Manual 953 (17th ed. 1999); List et al., J. Clin. Oncol. 8:1424 (1990).
MDS are grouped together because of the presence of dysplastic changes in one or more of the hematopoietic lineages including dysplastic changes in the myeloid, erythroid, and megakaryocytic series. These changes result in cytopenias in one or more of the three lineages. Patients afflicted with MDS may develop complications related to anemia, neutropenia (infections), and/or thrombocytopenia (bleeding). From about 10% to about 70% of patients with MDS may develop acute leukemia. In the early stages of MDS, the main cause of cytopenias is increased programmed cell death (apoptosis). As the disease progresses and converts into leukemia, a proliferation of leukemic cells overwhelms the healthy marrow. The disease course differs, with some cases behaving as an indolent disease and others behaving aggressively with a very short clinical course that converts into an acute form of leukemia. The majority of people with higher risk MDS eventually experience bone marrow failure. Up to 50% of MDS patients succumb to complications, such as infection or bleeding, before progressing to AML.
Primary and secondary MDS are defined by taking into account patients' prior history: previous treatments with chemotherapy, radiotherapy or professional exposure to toxic substances are factors delineating secondary MDS (sMDS) from primary MDS. Cytogenetically, one difference between the two groups is the complexity of abnormal karyotypes; single chromosome aberrations are typical for primary MDS, while multiple changes are more frequently seen in secondary disorders. Some drugs may have specific targets such as hydroxyurea for 17p and topoisomerases inhibitors for 11q23 and 21q22. The genetic changes in the malignant cells of MDS result mainly in the loss of genetic material, including probable tumor suppressor genes.
An international group of hematologists, the French-American-British (FAB) Cooperative Group, classified MDS into five subgroups, differentiating them from acute myeloid leukemia. See, e.g., The Merck Manual 954 (17th ed. 1999); Bennett J. M., et al., Ann. Intern. Med., 103(4): 620-25 (1985); and Besa E. C., Med. Clin. North Am. 76(3): 599-617 (1992). An underlying trilineage dysplastic change in the bone marrow cells of the patients is found in all subtypes. Information is available regarding the pathobiology of MDS, certain MDS classification systems, and particular methods of treating and managing MDS. See, e.g., U.S. Pat. No. 7,189,740 (issued Mar. 13, 2007), which is incorporated by reference herein in its entirety.
Nucleoside analogs have been used clinically for the treatment of viral infections and cancer. Most nucleoside analogs are classified as anti-metabolites. After they enter the cell, nucleoside analogs are successively phosphorylated to nucleoside 5′-mono-phosphates, di-phosphates, and tri-phosphates.
5-Azacytidine (National Service Center designation NSC-102816; CAS Registry Number 320-67-2), also known as azacitidine, AZA, 4-amino-1-(3,4-dihydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-1H-[1,3,5]triazin-2-one, 4-amino-1-β-D-ribofuranosyl-1,3,5-triazin-2(1H)-one, or 4-amino-1-β-D-ribofuranosyl-S-triazin-2(1H)-one, is currently marketed as the drug product VIDAZA®. 5-Azacytidine is a nucleoside analog, more specifically a cytidine analog. 5-Azacytidine is an antagonist of its related natural nucleoside, cytidine. 5-Azacytidine is also an antagonist of deoxycytidine. A structural difference between 5-azacytidine and cytidine is the presence of a nitrogen at position 5 of the cytosine ring in place of a carbon. 5-Azacytidine may be defined as having the molecular formula C8H12N4O5, a molecular weight of about 244.2 grams per mole, and the following structure:

After its incorporation into replicating DNA, 5-azacytidine forms a covalent complex with DNA methyltransferases. DNA methyltransferases are responsible for de novo DNA methylation and for reproducing established methylation patterns in daughter DNA strands of replicating DNA. Inhibition of DNA methyltransferases by 5-azacytidine leads to DNA hypomethylation, thereby restoring normal functions to morphologically dysplastic, immature hematopoietic cells and cancer cells by re-expression of genes involved in normal cell cycle regulation, differentiation and death. The cytotoxic effects of 5-azacytidine cause the death of rapidly dividing cells, including cancer cells, that are no longer responsive to normal cell growth control mechanisms. 5-Azacytidine also incorporates into RNA. The cytotoxic effects of 5-azacytidine may result from multiple mechanisms, including inhibition of DNA, RNA and protein synthesis, incorporation into RNA and DNA, and activation of DNA damage pathways.
5-Azacytidine has been tested in clinical trials and showed significant anti-tumor activity, such as, for example, in the treatment of myelodysplastic syndromes (MDS). See, e.g., Aparicio et al., Curr. Opin. Invest. Drugs 3(4): 627-33 (2002). 5-Azacytidine has undergone NCI-sponsored trials for the treatment of MDS and has been approved for treating all FAB subtypes of MDS. See, e.g., Kornblith et al., J. Clin. Oncol. 20(10): 2441-52 (2002); Silverman et al., J. Clin. Oncol. 20(10): 2429-40 (2002). 5-Azacytidine may alter the natural course of MDS by diminishing the transformation to AML through its cytotoxic activity and its inhibition of DNA methyltransferase. In a Phase III study, 5-azacytidine administered subcutaneously significantly prolonged survival and time to AML transformation or death in subjects with higher-risk MDS. See, e.g., P. Fenaux et al., Lancet Oncol., 2009, 10(3):223-32; Silverman et al., Blood 106(11): Abstract 2526 (2005).
5-Azacytidine has been difficult to synthesize, particularly for manufacturing at large commercial scales, in part because of its instability in water. For example, the s-triazine ring of 5-azacytidine is prone to degradation in water. In aqueous solutions at neutral pH, hydration of the 5,6-imine double bond occurs rapidly, followed by bond cleavage to yield the formyl derivative, N-(formylamidino)-N′-β-D-ribofuranosylurea, which deformylates to give 1-β-D-ribofuranosyl-3-guanylurea irreversibly. See, e.g., J. A. Beisler, J. Med. Chem., 1978, 21(2):204-08; K. K. Chan et al., J. Pharm. Sci. 1979, 68(7):807-12. In addition, the hydrolytic degradation of 5-azacytidine was studied as a function of pH, temperature, and buffer concentration. See, e.g., R. E. Notari et al., Pharm. Sci. 1975, 64(7):1148-57. At pH 1, the main degradation products were 5-azacytosine and 5-azauracil from the hydrolysis of 5-azacytidine. The instability of 5-azacytidine in water presents a challenge for the isolation and purification of 5-azacytidine from a solvent system that contains water, such as solvent systems used during the work-up stage of a reaction.
5-Azacytidine was first prepared via a multi-step synthesis starting from peracetylated 1-glycosylisocyanate. See Piskala et al., Collect. Czech. Chem. Commun., 1964, 29:2060-76. This method involves a reactive starting material (isocyanate) with a controlled stereochemistry (1-β configuration), which is not suitable for the production of large scale batches of 5-azacytidine. Many other existing methods for preparing 5-azacytidine involve steps that are difficult to scale-up, or involve the use of expensive reagents. Other methods do not describe purification steps necessary to produce Active Pharmaceutical Ingredient (API) that meets the purity standards for human use, or give poor overall yields of the purified 5-azacytidine product.
U.S. Pat. No. 7,038,038, issued May 2, 2006, which is incorporated herein by reference in its entirety, describes a process for preparing 5-azacytidine, comprising, inter alia, the steps of coupling a silylated 5-azacytosine with a protected β-D-ribofuranose in the presence of a non-metallic Lewis acid, such as trimethylsilyl trifluoromethanesulfonate (TMS-triflate), and deprotecting the product to give 5-azacytidine.
Metallic Lewis acids, such as stannic chloride, are generally less expensive and more readily available than non-metallic Lewis acids, such as TMS-triflate. However, the use of metal-based reagents in the synthesis of API intended for human use generally requires appropriate purification steps to remove metal-based impurities in order to consistently control the levels of residual metals in the final API. For the production of a drug substance intended for use in humans, current Good Manufacturing Practices (cGMP) are applicable. Procedures need to be in place that can control the levels of impurities and ensure that API batches are produced which consistently meet their predetermined specifications. For example, in a GMP environment, it is not acceptable to have one batch having a heavy metal content that is within a desired specification, and then have a batch run under similar circumstances having heavy metal impurities well over the desired specification.
A great need remains for a process to prepare pure 5-azacytidine suitable for human use, particularly on a commercial scale, that is, inter alia, safe, scalable, efficient, economically viable, and/or having other potential advantages.
Citation of any references in this Section of the application is not to be construed as an admission that such reference is prior art to the present application.